ANALYTICAL CHEMISTRY
494
procedure used for an unknown. The latter procedure is preferable, for in this case the iodine standards and unknown receive identical treatments. The reference curve obtained in this manner will deviate from the one shown in Table I, depending on the amount of iodine in the digestion and distillation reagents. A new curve should be prepared with each new batch of chromium trioxide solution. However, this is not inconvenient, because 2 liters of.this reagent are enough for 200 determinations. If commercial chromium trioxide sufficiently low in iodine cannot be obtained, the procedure used by Matthews, Curtis, and Brode (2) for preparing this reagent is recommended.
The average error in 20 determinations (each performed in triplicate) was *0.02 microgram and was independent of the amount of iodine in the range of 0.10 to 5.00 micrograms, LITERATURE CITED
(1) Gross, W. G., Wood, L. K., and MoHargue, J. S., Bx.4~.CHHM., 20, 900 (1948). (2) Matthews, N. L., Curtis, G. M.,and Brode, W. R., IND. ENO. CHEM.,ANAL.ED., 10, 612 (1938). RECEIVED March 28, 1949, Work done in connection with a project of the Kentucky Agricultural Experiment Station and published by permission of the director.
Estimation of Vitamin A in the Presence of Interfering Materials W. A. MCGILLIVRAY Massey Agricultural College, University of New Zealand, Palmerton North, ,V. Z . S T H E estimation of vitamin A by measurement of the absorp-
I tion at 325 mp, a correction must usually be made forathenumber pres-
ence of other substances which absorb in this region, and of methods have been introduced to make allowance for this irrelevant absorption. Morton and Stubbs ( 1 ) have recently investigated the nature of the correction required for various fish oils and have introduced a correction procedure based on measurements of the extinction coefficients at Xmsx. and at two other wave lengths such that, for pure vitamin A, E is 8 / 7 of Em=. From the difference between these two extinctions and the extent to which r
I
0.3-
tors affect both the position of Amax. and the shape of the absorption curve of the vitamin. The application of the correction procedure to vitamin Aalcohol plus interfering materials in ethyl dcohol is a typical case and is illustrated in Figure 1. In the inset graph the dotted line represents an idealized curve for the interfering substances which together with the vitamin present give the a b sorption curve for the solution shown as a full line. Readings, giving extinction coefficients denoted by the letters A , B , and C, respectively, are taken at wave lengths 325,310, and 340 mp-Le., A,,., Amax. - 15 mp, and . , , ,A 15 mp. The absorption due to the pure vitamin A present at these three wave lengths is A - (z y) at 325 mp, B - (z 2y) at 310 mp, and C - z a t 340 mp. For pure vitamin h alcohol in ethyl alcohol the extinction ratios E3,0/E326 mp and E340/E325mp have been calculated in this laboratory as 0.846 and 0.771, rpspectively. It follows therefore that:
+
+
B Q r-
50.2 -
,
+
- (X + 2 y ) = 0.846 [ A - (Z + y ) ] C - z = 0.771 [ A - (X + y ) l
(1) (2)
Adding Equations 1 and 2 gives thr expression
+ C - 2 (Z + y) = 1.617 [A - + y ) ] (3) which in terms of z + w, the irrelevant absorption at 325 mp, reduces to x + y = 2.60 (B + C - 1.617 A ) (4)
I
B
V
+
m 0.1
0.2
0.6 INTERFERENCE ( X + Y )
0.4
0.8
Figure 1. Application of Correction Procedure
(2
The actual amount of this irrelevant absorption may conveniently be read directly from a graph as shown in Figure 1 and subtracted from reading A to give the extinction due to vitamin -4. Alternatively, Equation 4 may be rewritten to give A - (z y), the extinctioii due to the vitamin A present, directly
+
A they deviate from 6 / ~ of Emax.,the irrelevant absorption a t Am*.. may be calculated. A similar method, but one which lends itself to a simpler calculation, has been in use for some time in this laboratory for detecting and estimating vitamin A in the presence of partially oxidized carotene. This method, in which readings are taken at Amax. and a t two other wave lengths equidistant above and below it, is capable of extension to other examples of superimposed irrelevant absorption, provided the points corresponding to the three wave lengths on the curve for the interfering substances alone lie in a straight line. Over the small wave band considered, this generally applies and the proviso is common to all such three-point methods. The wave lengths a t which readings are taken and the interpretation of these readings will depend on the solvent employed and on whether the vitamin is free or esterified, inasmuch as these fac-
- (Z + y)
=
2.60 [2A
- (B + C ) ]
(325 mp) for vitamin A alcohol in ethyl ahand taking hol as 1780 this becomes Concentration of vitamin A in test solution = 14.6 [2A Equations similar to these but with new constailts can be derived for other solvents snd for the vitamin in esterified forms. Although a graphical solution could be applied to Morton and Stubbs’s method, it is considered that the selection of wave lengths equidistant from Amax. in the present method renders it simpler and therefore more readily applicable to routine work. LITERATURE CITED
(1) Morton, R. A., and Stubbs,A. L., Biochem. J.,42, 195 (1948) RECEIVED December 10, 1948.
